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OR I G I N A L R E S E A R CH
Nutrient sink limitation constrains growth in two barleyspecies with contrasting growth strategies
Angela C. Burnett1 | Alistair Rogers2 | Mark Rees1 | Colin P. Osborne1
and 19.27 μM sodium chloride, in Reverse Osmosis water. Long Ash-
ton's solution provides mineral nutrients in proportions appropriate
to the requirements of the plant, so the treatment altered the
application of all mineral nutrients in accordance with these propor-
tions. Plants did not display visible signs of mineral deficiency or tox-
icity (Supporting Information Figure S1).
2.2 | 15N uptake
Nitrate labelled with the 15N stable isotope (15NO3−) was fed 24 hr
before the final harvest at 42 days after germination. A 15N‐labelledpotassium nitrate and calcium nitrate (10% labelled atom) were sub-
stituted for the same mass of unlabelled potassium nitrate and cal-
cium nitrate in the Long Ashton nutrient solution, such that the
plants received their usual dose of all nutrients.
A subsample of the ground, freeze‐dried material obtained for
metabolite measurements (see below) was analysed for 15N enrich-
ment using an ANCA GSL 20‐20 Mass Spectrometer (Sercon PDZ
Europa, Crewe, UK). Enrichment was calculated as 15N/(14N + 15N)
and adjusted for percentage labelled atom fed, and values from con-
trol samples which did not receive 15N were subtracted from these
data in order to give 15N enrichment relative to the baseline level
for each organ type and nutrient treatment (baseline 15N of control
samples did not differ between the two species).
2.3 | Growth and allocation
Shoot area was obtained by photographing plants twice per week
starting 8 days after germination, using the method described by
Burnett et al. (2016). For very young plants, only one photograph
was required, whereas six photographs from evenly spaced angles
were used for larger plants. Shoot area was calibrated with whole‐plant dry mass using a batch of 29 additional plants, not used in the
main study, which were also photographed twice weekly. These
additional individuals were harvested and oven‐dried 12, 19, 26, 33,
and 40 days after germination in order to calibrate shoot area to leaf
area and dry mass (r2 = 0.88), and to analyse partitioning to different
organs. Biomass data for plants in the main study were predicted
using this calibration. Mass‐based relative growth rate (RGR) was
obtained for each individual in the main study by converting non‐destructive projected area measurements to whole‐plant dry mass
using the calibration from the additional batch of destructively har-
vested plants and calculating the change in the natural logarithm of
dry mass over time. Tillers were counted on all plants in the main
study immediately prior to metabolite harvests.
Dry mass fractions of leaf, leaf sheath, and root were calculated
for each oven‐dried individual from the additional batch of plants
and used to calculate leaf mass ratio (LMR; kg leaf dry mass per kg
plant dry mass). Mean LMR values for each species and nitrogen
concentration were then used to predict LMR for plants in the main
study. Specific leaf area (SLA; m2 leaf per kg leaf) was recorded for
all plants in the main study by measuring the area and fresh mass of
harvested leaves, and converting fresh mass into dry mass using the
calibration obtained from oven‐dried plants. Leaf area ratio (LAR; m2
leaf per kg plant) for each individual in the main study was obtained
by multiplying SLA by predicted LMR.
BURNETT ET AL. | 3
Data from the additional subset of 29 destructively harvested
and oven‐dried plants were used only for LMR (Table 1) and for cali-
brating RGR and LMR for plants in the main study. All other data
were obtained from the plants in the main study.
2.4 | Metabolites
Metabolite harvests were carried out on plants from the main study.
Harvests were carried out at 14, 28, and 42 days after germination;
all these timepoints were within the vegetative growth stage. Plants
were always watered 24 hr before harvest so that all samples would
be equivalent with respect to watering schedule. For each nutrient
level and species, one replicate from each of the three growth cham-
bers was taken, giving a total of 18 plants at each timepoint. Due
to the small size of plants harvested 14 days after germination, it
was necessary to pool two individuals of the same species and nutri-
ent treatment for each replicate, to obtain enough material for analy-
sis. Plants were harvested within 1 hr before dawn, in order to
capture pre‐dawn metabolite levels, when the diurnal concentrations
of carbohydrate are expected to be minimal, following previous work
(Burnett et al., 2016). Plants were separated into leaf, leaf sheath,
and root, flash‐frozen in liquid nitrogen and stored at −80°C; sam-
ples were subsequently freeze‐dried prior to analysis. For small
plants, the entire plant was harvested; for larger plants, representa-
tive samples of each organ from both young and old tissue were
harvested. Metabolite assays were carried out as described previ-
ously (Burnett et al., 2016): glucose, fructose, sucrose, low and high
degree of polymerisation (LDP and HDP) fructans, and starch were
quantified using continuous enzymatic substrate assays, whilst
nitrate, amino acids, and proteins were quantified using the Griess
reaction, fluorescamine assay, and bicinchoninic acid assay, respec-
tively. Elemental CHN was analysed using a 2400 Series II CHN
analyser (PerkinElmer, Waltham, MA, USA). More samples were
available for CHN analysis than for metabolite assays, due to the
small size of some plants. Metabolites and CHN were expressed per
gram carbohydrate‐corrected dry weight (CCDW), obtained by sub-
tracting the total mass of non‐structural carbohydrate (the sum of:
glucose, fructose, sucrose, LDP and HDP fructan, and starch) from
the dry mass of each sample. Metabolite data are available in Sup-
porting Information Table 1.
2.5 | Statistical methods
All data were analysed in R (R Core Team, 2015) using Type II
ANOVA. Natural logarithmic transformations were performed prior
to analysis to satisfy the normality assumptions of ANOVA.
3 | RESULTS
Net nitrogen uptake (Figure 1) decreases significantly as nutrient
treatment is lowered (F2,10 = 54.6, p < 0.001), but there is no signifi-
cant species effect (either main effect or interaction, p > 0.5 in each
case) on net nitrogen uptake (Figure 1). Relative growth rate (RGR;
Figure 2) is consistently higher in annual than in perennial barley,
suggesting that the annual invests more resources into growth than
the perennial, even at low nutrient levels (significant effect of spe-
cies: F1,11 = 9.77, p < 0.01). This shows that the nutrient use effi-
ciency of growth is higher in the annual. RGR decreases significantly
in annual and perennial barley when nutrient treatment is lowered
TABLE 1 Root:shoot, leaf mass, sheath mass, and root massratios in annual and perennial barley at different nutrient treatmentlevels
SpeciesLownutrients
Mediumnutrients
Highnutrients
Root:shoot
ratio
Annual 1.60 ± 0.2 0.80 ± 0.2 0.54 ± 0.1
Perennial 1.54 ± 0.3 0.83 ± 0.2 0.59 ± 0.07
Leaf mass
ratio
Annual 0.33 ± 0.03 0.42 ± 0.03 0.46 ± 0.01
Perennial 0.27 ± 0.01 0.35 ± 0.01 0.44 ± 0.04
Sheath mass
ratio
Annual 0.07 ± 0.004 0.17 ± 0.03 0.24 ± 0.02
Perennial 0.14 ± 0.05 0.20 ± 0.07 0.19 ± 0.02
Root mass
ratio
Annual 0.60 ± 0.03 0.41 ± 0.06 0.30 ± 0.01
Perennial 0.59 ± 0.05 0.45 ± 0.05 0.37 ± 0.03
Allocation to roots increases at lower nutrient supplies (p < 0.001) and
allocation to leaves and sheaths decreases at lower nutrient supplies
(p < 0.001). These data are obtained from the subset of 29 additional
plants harvested for biomass calibration described in the Materials and
Methods section. For root:shoot ratio, root mass is divided by the sum
of leaf and sheath mass for each individual and the results averaged. For
leaf, sheath, and root mass ratios, the ratio is the dry mass of that organ
divided by the dry mass of the whole plant. 18 annuals and 11 perennials
were harvested at five timepoints. Data show mean ± SE (at low, med-
ium, high nutrients, annual n = 6, 6, 5; perennial n = 4, 2, 5). For brevity,
age effects have not been included in this summary, but they are dis-
cussed elsewhere in the manuscript.
F IGURE 1 Net nitrogen uptake rate of annual (filled circles, solidline) and perennial barley (hollow circles, dashed line) decreases asnutrient treatment level is lowered (p < 0.001). Uptake wasmeasured over 24 hr before harvesting plants 42 days aftergermination. Data show mean ± SE (n = 3)
4 | BURNETT ET AL.
(F2,11 = 7.98, p < 0.01), and this effect is especially strong when
nutrients are decreased from the medium to low treatment level.
Biomass partitioning is also affected by nutrient treatment. In
accordance with the change in RGR, tillering decreases substantially
at low nutrient levels (Figure 3); this effect is greater in older plants
(significant nutrient × age interaction for tillering: F4,24 = 13.1,
p < 0.001). However, whilst the nutrient effect is strong, there is
neither a difference between species nor a nutrient × species inter-
action (p > 0.6 in each case). Allocation to roots (Table 1) increases
at lower nutrient supplies (for root:shoot ratio, F1,19 = 40.6,
p < 0.001; for root mass ratio (RMR), F1,19 = 38.9, p < 0.001), but
does not differ between species. In both species, sheath mass ratio
and leaf mass ratio (SMR and LMR; Table 1) decrease at low nutrient
levels (for sheaths, F1,19 = 25.9, p < 0.001; for leaves, F1,19 = 30.1,
p < 0.001). The annual allocates more biomass to roots earlier in
development and more to sheaths later in development whilst the
opposite trend is seen in the perennial (significant species × age
interactions: for SMR, F1,19 = 16.8, p < 0.001; for root:shoot ratio,
F1,19 = 10.6, p < 0.01).
Leaf area ratio (LAR) does not differ between species, although
specific leaf area (SLA) is slightly higher in the perennial (Table 2;
SLA: F1,45 = 5.97, p < 0.05). LAR and SLA decrease with age in both
species (LAR: F1,45 = 34.6, p < 0.001; SLA: F1,45 = 42.2, p < 0.001),
and LAR decreases at lower nutrient treatment levels (F1,45 = 56.6,
p < 0.001), in addition to the decrease in LMR documented above
(Tables 1 and 2). Thus at lower nutrient treatment levels, proportion-
ately less biomass is allocated to leaves, and there is a lower leaf
area per unit of plant biomass.
Annual plants have a greater absolute size than perennials
(Table 3). Predicted biomass increases with age, especially in annuals
(species × age interaction: F1,415 = 25.1, p < 0.001) and especially at
higher nutrient levels (nutrient × age interaction: F2,415 = 48.3,
p < 0.001). For brevity, only predicted biomass data collected before
each destructive harvest are presented in Table 3, but the effects
reported here are for all predicted biomass data (obtained twice
weekly).
F IGURE 2 Relative growth rate (RGR, g g−1 day−1) is higher inannual (solid line) than in perennial (dashed line) barley (p < 0.01).Data are from plants harvested 28 and 42 days after germination.Data show mean ± SE (for annuals, n = 5, 6, 4, and for perennials,n = 1, 3, 3 at low, medium, and high nutrients, respectively)
F IGURE 3 Tillering increases with nutrient treatment and thiseffect increases with age in both annual (filled circles, solid line) andperennial (hollow circles, dashed line) barley (p < 0.001). Data showmean ± SE (n = 3), for plants harvested 14, 28, and 42 days aftergermination; at all three ages, plants at low nutrient treatment haveonly one tiller. Points are offset with respect to x‐axis position, toincrease readability
TABLE 2 Leaf area ratio (LAR) and specific leaf area (SLA) inannual and perennial barley grown at different nutrient levels andharvested 14, 28, and 42 days after germination
SpeciesLownutrients
Mediumnutrients
Highnutrients
LAR at
14 days
Annual 11.3 ± 0.5 14.7 ± 1 20.0 ± 2
Perennial 9.5 ± 0.8 16.1 ± 0.7 19.9 ± 2
LAR at
28 days
Annual 12.0 ± 0.2 13.7 ± 0.5 17.3 ± 1
Perennial 12.3 ± 3 13.6 ± 1 16.1 ± 2
LAR at
42 days
Annual 9.8 ± 0.1 10.3 ± 0.6 12.3 ± 0.2
Perennial 7.7 ± 0.4 10.6 ± 1 13.2 ± 0.3
SLA at
14 days
Annual 34.3 ± 1 35.0 ± 3 43.4 ± 4
Perennial 35.1 ± 3 46.1 ± 2 45.3 ± 4
SLA at
28 days
Annual 36.5 ± 0.7 32.6 ± 1 37.6 ± 2
Perennial 45.7 ± 12 38.9 ± 3 36.6 ± 4
SLA at
42 days
Annual 29.6 ± 0.3 24.6 ± 1 26.8 ± 0.4
Perennial 28.6 ± 2 30.3 ± 3 30.1 ± 0.7
LAR decreases as nutrient treatment level is lowered (p < 0.001). SLA is
higher in perennial than annual barley overall (p < 0.05). Both LAR and
SLA are lower in older plants (p < 0.001). These data are for plants in
the main study. LAR is expressed here as m2 leaf/kg plant and SLA is
expressed here as m2 leaf/kg leaf. LAR is the product of SLA (measured
on all plants in the main study at the time of metabolite harvest) and
LMR (from the subset of plants harvested for biomass calibration; see
Table 1). Data show mean ± SE (n = 3).
BURNETT ET AL. | 5
Overall, a decrease in nutrient treatment leads to a decrease in
the concentration of nitrogen metabolites in annual and perennial
barley (Figure 4), especially when nutrient treatment is decreased
from medium to low. A comparatively smaller decrease is observed
between high and medium nutrient levels, similar to the response of
RGR to nutrient treatment (Figures 2 and 4). Nitrate and amino acids
generally show a response to nutrient treatment level across the
plant. In annual and perennial barley, with decreasing nutrient treat-
ment, there is a significantly lower nitrate concentration in leaf
(F2,24 = 64.5, p < 0.001), leaf sheath (F1,11 = 17.9, p < 0.01), and
root (F2,25 = 182.7, p < 0.001). Nitrate in the sheath tissue is signifi-
cantly higher in the annual (F1,11 = 45.9, p < 0.001). Amino acid con-
centration in leaf and root also decreases at lower nutrient
treatment levels, and this effect is greater in older plants (nutrient
treatment × age interaction in leaf: F4,24 = 12.1, p < 0.001; in root:
F4,25 = 7.9, p < 0.001). Amino acid concentration in the sheath is
not affected by nutrient treatment, but increases with age (Fig-
ure 4d,e; F1,11 = 20.8, p < 0.001). In the root, amino acid content is
higher in perennial than annual barley (F1,24 = 26.1, p < 0.001). In
contrast to nitrate and amino acids, protein does not show a whole‐plant response to nutrient treatment level. Protein concentration
shows an overall decrease from high to low nutrient treatment levels
in the leaf (Figure 4; F2,25 = 13.9, p < 0.001), but does not show a
significant change in sheath or root.
TNC in the leaf is significantly higher in the annual (F1,24 = 14.8,
p < 0.001), as would be expected if the annual is more carbon sink
limited than the perennial. Leaf TNC is also affected by nutrient
treatment (F2,24 = 6.1, p < 0.01): leaf TNC increases when nutrient
treatment is lowered from medium to low in younger plants (Fig-
ure 5a), yet decreases at low nutrient levels in older plants (Fig-
ure 5b,c). However, contrary to expectations that carbon source
limitation would increase at low nutrient levels, leaf TNC increases
when nutrient treatment is lowered from high to medium, that is,
leaf TNC accumulates more at medium nutrient treatment levels
than at high levels. This indicates that carbon source limitation does
not increase when nutrient treatment is decreased. Rather, carbon
source limitation is greater at high nutrient treatment levels. Root
TNC shows a significant nutrient treatment × age interaction
(F4,25 = 9.3, p < 0.001): the increase in TNC at lower nutrient treat-
ment is most pronounced 14 days after germination (Figure 5f).
Elemental N content (Figure 6a–c) decreases at lower nutrient
treatment level in leaf (nutrient treatment × age interaction:
Predicted biomass increases with age especially in annual barley, and
especially at higher nutrient treatment levels (p < 0.001 for each of these
interactions). These data are for plants in the main study. Biomass (g) is
predicted from leaf area data, using the correlation obtained for the sub-
set of additional plants harvested for biomass calibration. Predicted bio-
mass is derived from photographs preceding destructive harvests (carried
out at 14, 28, and 42 days after germination). Data show mean ± SE,
with n decreasing over time due to destructive harvests (at 12 days, at
low, medium, high nutrients, annual n = 17, 18, 18; perennial n = 7, 13,
15; at 22 days, at low, medium, high nutrients, annual n = 10, 10, 10;
perennial n = 6, 6, 9; at 40 days, at low, medium, high nutrients, annual
n = 5, 5, 5; perennial n = 3, 3, 4).
6 | BURNETT ET AL.
nutrient level. However, this is not always the case (Figure 5), even
though leaf protein shows a small increase in the higher nutrient
treatment levels (Figure 4), indicating that carbon source limitation is
not alleviated by nutrients. In addition, amino acid:sucrose ratio,
another indicator of carbon source limitation, increases in the root at
higher nutrient treatment levels, further indicating that carbon
source limitation increases at high nutrient levels rather than
decreasing (Figure 7b). Consistent with this hypothesis, carbon
becomes an increasingly limiting resource at higher nutrient treat-
ment levels, and the leaf and sheath mass ratios in both species
increase to compensate for this effect (Table 1). Taken together, this
evidence shows that the nutrient limitation on growth is mediated
by a direct constraint on expansion growth (hypothesis (2)) rather
than acting via carbon limitation (hypothesis (1)).
Regarding hypothesis (3), the investment of nitrogen into growth
and storage shows large decreases when nutrient treatment is
decreased from a medium to a low supply, but this response is smal-
ler when nutrients are decreased from a high to a medium nutrient
supply, especially in annual barley. These results indicate a significant
nutrient source limitation at low nutrient levels, but a nutrient sink
limitation at high nutrient levels (Figures 2, 4 and 6). The relatively
small difference in nutrient investment between medium and high
nutrient levels shows that plants are nutrient sink limited under
medium nutrient treatment levels. Although the annual invests more
resources in overall growth (Figure 2, Table 3), the perennial is better
able to increase investment in growth under high nutrient levels, in rel-
ative terms, despite its smaller size (Figure 2). This result suggests that
growth in the perennial is less limited by sink development than
Nutrients (% Long Ashton’s)
Nutrients (% Long Ashton s) Nutrients (% Long Ashton s) (% Long Ashton s) Nutrients
Nutrients (% Long Ashton s) (% Long Ashton s) Nutrients
Nutrients (% Long Ashton s) (% Long Ashton s) Nutrients
(a) (b) (c)
(f) (g) (h)
(d) (e)
F IGURE 4 Partitioning of nitrogen resources in different organs of annual (filled symbols, solid line) and perennial (hollow symbols, dashedline) barley. Statistics are outlined in the main text. Panel shows allocation of nitrogen to protein (filled and hollow circles), free nitrate (filledand hollow squares), and free amino acids (filled and hollow triangles). All data are expressed in mmol N per gram carbohydrate‐corrected dryweight (CCDW). CCDW is obtained by subtracting the mass of total non‐structural carbohydrate (the sum of: glucose, fructose, sucrose, LDPand HDP fructan, and starch) from the dry mass of each sample. (a) Leaf 14 days after germination; (b) leaf 28 days; (c) leaf 42 days; (d) leafsheath 28 days; (e) leaf sheath 42 days; (f) root 14 days; (g) root 28 days; (h), root 42 days. Leaf and leaf sheath tissues were pooled for theharvests at 14 days. Data show mean ± SE (n = 3). Points are offset with respect to x‐axis position, to increase readability
BURNETT ET AL. | 7
growth in the annual, so improving nutrient sink strength will be
important for improving the growth and yield of annual barley.
4.1 | Perennial barley shows a greater growthresponse to nutrients
Annual and perennial barley have the same net nitrogen uptake rate
(Figure 1), and both show a substantial decrease in nitrogen uptake
with decreasing nutrient treatment level (Figure 1), consistent with
expectations. As the nutrient treatment decreases from medium to
low nutrient supply, plants show large growth and storage responses,
with both the amount of plant material (Figure 2) and the concentra-
tion of nitrogen (Figure 4) decreasing significantly.
Annual barley grows faster overall (Figure 2) and is larger than
the perennial (Table 3) indicating higher nutrient use efficiency of
growth. RGR is more strongly limited by nutrient supply in the
perennial, suggesting that the lower nutrient efficiency of growth in
the perennial leads to stronger nutrient limitation when nutrient sup-
ply is restricted. The perennial shows a relatively greater growth
response to nutrient level than the annual (Figure 2) and has a
higher leaf nitrogen concentration (Figure 6a). This suggests that,
under nitrogen‐limited conditions (i.e. the low nutrient treatment),
the perennial preferentially allocates nitrogen to storage rather than
growth, consistent with the hypothesis that the perennial will con-
serve mineral nutrients (Campbell & Grime, 1992). However, con-
trary to the hypothesis that growth in the perennial would be more
Nutrients (% Long Ashton’s) Nutrients (% Long Ashton’s) Nutrients (% Long Ashton’s)
Nutrients (% Long Ashton’s) Nutrients (% Long Ashton’s)
Nutrients (% Long Ashton’s) Nutrients (% Long Ashton’s) Nutrients (% Long Ashton’s)
(a) (b) (c)
(f) (g) (h)
(d) (e)
F IGURE 5 Partitioning of total non‐structural carbohydrates (TNC) in different organs of annual (filled symbols, solid line) and perennial(hollow symbols, dashed line) barley. Statistics are outlined in the main text. Panel shows TNC (filled and hollow circles) and the proportion ofTNC allocated to fructans (filled and hollow triangles). TNC is the sum of glucose, fructose, sucrose, and LDP and HDP fructans. All data areexpressed in mmol C per gram carbohydrate‐corrected dry weight (CCDW). CCDW is obtained by subtracting the mass of total non‐structuralcarbohydrate (the sum of: glucose, fructose, sucrose, LDP and HDP fructan, and starch) from the dry mass of each sample. (a) Leaf 14 daysafter germination; (b) leaf 28 days; (c) leaf 42 days; (d) leaf sheath 28 days; (e) leaf sheath 42 days; (f) root 14 days; (g) root 28 days; (h) root42 days. Leaf and leaf sheath tissues were pooled for the harvests at 14 days. Data show mean ± SE (n = 3). Points are offset with respect tox‐axis position, to increase readability
8 | BURNETT ET AL.
nutrient sink limited and growth in the annual more nutrient source
limited, the perennial shows a greater response to increased nutrient
treatment. This indicates that growth in the perennial is in fact less
nutrient sink limited than growth in the annual.
Contrary to expectations, the perennial has a higher SLA (Table 2)
and higher leaf nitrogen concentration (Figure 6) than the annual—traits that are generally associated with fast‐growing species in the
ecological literature (Reich et al., 2003; Wright et al., 2004). This is
consistent with previous work on this species (Burnett et al., 2016),
which showed carbon source limitation of growth in the perennial,
since it is investing in carbon acquisition by the leaves in order to
match carbon and nitrogen supply. Indeed, the higher SLA and leaf
nitrogen concentration observed here for the perennial indicate that it
may have the potential to be a rather fast‐growing species despite its
perennial life history strategy. Potential RGR has previously been cor-
related with nitrogen uptake capacity (Garnier et al., 1989) and here
the net nitrogen uptake rates are very similar for annual and perennial
barley, although the perennial never matches the RGR of the annual.
RGR (Figure 2) is higher in the annual, but LAR (the product of SLA
and LMR) does not differ between species (Table 2). Whilst SLA tends
to be the major contributor to LAR and thus RGR in herbaceous spe-
cies (Poorter & van der Werf, 1998), some studies have found that
RGR correlates with LMR rather than SLA (references within Garnier,
1992), which is consistent with these data. Furthermore, since RGR is
the product of net assimilation rate (NAR) and leaf area ratio (LAR;
Poorter & Remkes, 1990), the higher RGR of annual compared to
perennial barley seen here is likely due to higher photosynthetic assim-
ilation, as seen in previous work with this species (Burnett et al.,
2016). Two further points of contention are: whether RGR regulates
resource uptake or whether resource uptake is regulated by RGR (Gar-
nier et al., 1989; Rodgers & Barneix, 1988); and the extent to which
uptake is regulated by demand (Taulemesse, Le Gouis, Gouache,
Gibon, & Allard, 2015). Indeed, nitrate itself is an important regulator
of nitrogen uptake (Masclaux‐Daubresse et al., 2010). In addition to
elucidating the relative contribution of nutrient source and sink to
growth, a deeper understanding of the molecular drivers that underpin
regulation of nutrient uptake will be an important component of
improving crop nutrient use efficiency.
F IGURE 6 Total nitrogen concentrationand carbon:nitrogen (C:N) ratio in annual(filled circles, solid line) and perennial(hollow circles, dashed line) barley.Statistics are outlined in the main text.Carbohydrate‐corrected dry weight(CCDW) was used for these elemental Nand C concentrations. CCDW is obtainedby subtracting the mass of total non‐structural carbohydrate (the sum of:glucose, fructose, sucrose, LDP and HDPfructan, and starch) from the dry mass ofeach sample. (a) Leaf nitrogen; (b) sheathnitrogen; (c) root nitrogen; (d) leaf C:Nratio; (e) sheath C:N ratio; (f) root C:Nratio. Data show mean across all ages (14,28, 42 days after germination) due to lackof significant age effects ± SE (at low,medium, high nutrients, annual n = 9, 9, 9in leaf and root and 1, 6, 6 for sheath;perennial n = 4, 7, 9 for leaf, 0, 5, 5 forsheath, and 5, 7, 9 for root). There aremore samples for CHN analysis than formetabolite analysis due to the small size ofsome samples. Points are offset withrespect to x‐axis position, to increasereadability
BURNETT ET AL. | 9
4.2 | Annual and perennial barley do not storeexcess nitrogen as protein
Nitrate is a labile store and therefore shows a particularly strong
response to nutrient treatment (Figure 4); this metabolite also shows
a strong response to nitrogen treatment in wheat (Devienne, Mary,
& Lamaze, 1994) and Arabidopsis (Tschoep et al., 2009) and, like pro-
tein, constitutes a key store for nitrogen in herbaceous plants (Mil-
lard, 1988). Here, nitrogen is taken up from the soil as nitrate, which
is readily accumulated in plant tissues without the energetic cost or
carbon skeletons needed to reduce nitrate to amino acids. Accumu-
lating nitrate therefore means that nitrogen storage can increase
under high nutrient treatment levels, even if carbon is limiting. Such
carbon limitation could arise quickly in young plants. Consistent with
this idea, amino acid accumulation is greater in older plants (Fig-
ure 4). Compared to nitrate and amino acids, protein concentration
remains relatively constant at all nutrient treatment levels, except in
leaf tissue (Figure 4) where a small decrease occurs when nutrient
treatment level decreases. Excess nitrogen is thus preferentially
stored as nitrate (short term storage) or amino acids (intermediate
storage), or invested in growth, rather than being used to elevate
whole‐plant protein concentration. The lack of an ontogenetic effect
on leaf protein during vegetative growth is also notable (Figure 4):
rather than regulating leaf protein concentration as leaves get older,
barley plants create more leaf tissue and maintain the same protein
concentrations; this contradicts the way in which many earth system
models deal with nutrient limitation and is therefore an important
avenue for further investigation.
4.3 | High nutrient levels increase carbon sourcelimitation
Storage of nitrogen as nitrate and amino acids suggests that plants
are carbon source limited as protein synthesis requires additional
carbon. Both species show a decrease in N concentration and an
increase in C:N ratio as the nutrient treatment level is lowered (Fig-
ure 6). This corresponds with a shift from excess nitrogen to excess
carbon in the plants (Stitt & Krapp, 1999). However, this effect is
observed when nutrient treatment is decreased from medium to low,
but not between high and medium nutrient levels (Figure 6) despite
a high nitrate availability and uptake rate, suggesting that plants are
reaching their maximum capacity for nitrogen storage at high nutri-
ent treatment levels, and are carbon source limited. Leaf TNC data
(Figure 5) also show carbon source limitation at high nutrient levels.
The higher LMR and SMR in both species at higher nutrient
treatment levels (Table 1) enables greater acquisition of carbon,
which becomes an increasingly limiting resource at higher nutrient
treatment levels; conversely, more biomass is allocated to roots in
low nutrient environments (Aerts & Chapin, 2000). Not only does
LMR increase with nutrient treatment in both species, but LAR also
increases (Tables 1 and 2), as observed by Garnier et al. (1989),
enabling greater photosynthetic carbon acquisition since there is a
greater, thinner leaf area for light harvesting. This suggests an
increase in carbon source limitation at high nutrient levels. Both spe-
cies show an increase in tillering as nutrient treatment is increased
(Figure 3), as observed in wheat by Taulemesse et al. (2015), and
especially when plants are older, facilitating a rapid increase in allo-
cation to shoots and thus enabling greater carbon acquisition.
In addition to these structural changes, and the differences in
elemental carbon and nitrogen concentrations, root amino acid:su-
crose ratio increases at higher nutrient treatment levels (Figure 7b).
This indicates that carbon source limitation is increasing, since the
available amino acids outsupply the corresponding supply of avail-
able carbon necessary to fuel growth (Isopp, Frehner, Long, & Nös-
berger, 2000; Paul & Driscoll, 1997; Stitt & Krapp, 1999). The
increase is particularly pronounced between low and medium
F IGURE 7 Amino acid:sucrose ratio is higher in perennial (hollowcircles, dashed line) than in annual (filled circles, solid line) barley inboth (a) leaf and (b) root (p < 0.001). In the root, amino acid:sucroseratio increases with increasing nutrient treatment level (p < 0.001).Data show mean across all ages (14, 28, 42 days aftergermination) ± SE (in leaf at low, medium, high nutrients, annualn = 6, 8, 9, perennial n = 9, 8, 9; in root at low, medium, highnutrients, annual n = 2, 7, 8, perennial n = 3, 6, 7). Insufficient datawere available for sheath
10 | BURNETT ET AL.
nutrient treatment levels. The higher amino acid:sucrose ratio in the
perennial, and lower TNC concentration indicates that the perennial
is more carbon source limited than the annual (Figures 5–7); this cor-
roborates the evidence from previous work on these species (Bur-
nett et al., 2016).
4.4 | Annual barley is more nutrient sink limitedthan perennial barley at moderate nutrient supply
In addition to the differences in the nutrient responses of each spe-
cies, there are broad similarities revealed by the nutrient treatment.
The treatment conditions are sufficiently strong that, at low nutrient
treatment, growth in both annual and perennial barley is strongly
nutrient source limited, as shown by high C:N ratios (Figure 6d–f),low nutrient uptake rates (Figure 1) and low growth rates compared
to medium nutrient levels (Figure 2). At high nutrient levels, growth
in the annual is more nutrient sink limited than the perennial, shown
by its lower relative ability to increase growth (Figure 2). Growth at
the medium nutrient supply appears to be more nutrient sink limited
in annual than perennial barley, since increasing the nutrient level
further has a much greater effect in the perennial.
In general, the higher RGR of annual plants arises from their
large investment in leaf area and photosynthetic capacity: specific
leaf area (SLA, mm2 leaf per gram leaf), nitrogen content, and parti-
tioning of that nitrogen to the photosynthetic machinery are higher
in the leaves of annuals, enabling greater carbon acquisition due to a
larger light‐harvesting area per unit leaf mass and a greater nitrogen
density associated with the photosynthetic machinery, thus facilitat-
ing high rates of carbon assimilation and faster growth (Garnier &
White, A. C., Rogers, A., Rees, M., & Osborne, C. P. (2016). How can we
make plants grow faster? A source–sink perspective on growth rate.
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gers, F., … Villar, R. (2004). The worldwide leaf economics spectrum.
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
How to cite this article: Burnett AC, Rogers A, Rees M,
Osborne CP. Nutrient sink limitation constrains growth in two
barley species with contrasting growth strategies. Plant Direct.